A microchip reactor coated with a gold nanoparticle network entrapping trypsin has been designed for the efficient on-line proteolysis of low-level proteins and complex extracts originating from the mouse macrophages. The nano-structured surface coating was assembled via a layer-by-layer electrostatic binding of poly(diallyldimethylammonium chloride) and gold nanoparticles (AuNPs). The assembly process was monitored by UV-vis spectroscopy, atomic force microscopy and quartz-crystal microbalance. The controlled adsorption of trypsin was theoretically studied on the basis of the Langmuir isotherm model, and the fitted Gmax and K values were estimated to be 1.2×10–7 mol/m2 and 4.1×105 M–1, respectively. Enzymatic kinetics assay confirmed that trypsin, which was entrapped in the biocompatible AuNPs network with a high loading capacity, preserved its bioactivity. The maximum proteolytic rate of the adsorbed trypsin was 400 mM/(min·µg). Trace amounts of proteins down to femtomole per analysis were digested using the microchip reactor and the resulting tryptic products were identified by MALDI-TOF MS/MS. The protein mixtures extracted from the mouse macrophages were efficiently identified by on-line digestion and LC-ESI-MS/MS analysis.